Specific FRET Probes Sensitive to Chitosan-Based Polymeric Micelles Formation, Drug-Loading, and Fine Structural Features

Abstract

Förster resonance energy transfer (FRET) probes are a promising tool for studying numerous biochemical processes. In this paper, we show the application of the FRET phenomenon to observe the micelle formation from surfactants, micelles self-assembling from chitosan grafted with fatty acid (oleic—OA, or lipoic—LA), cross-linking of SH groups in the micelle’s core, and inclusion and release of the model drug cargo from the micelles. Using the carbodiimide approach, amphiphilic chitosan-based polymers with (1) SH groups, (2) crosslinked with S-S between polymer chains, and (3) without SH and S-S groups were synthesized, followed by characterization by FTIR and NMR spectroscopy. Two pairs of fluorophores were investigated: 4-methylumbelliferon-trimethylammoniocinnamate—rhodamine (MUTMAC–R6G) and fluorescein isothiocyanate—rhodamine (FITC–R6G). While FITC–R6G has been described before as an FRET-producing pair, for MUTMAC–R6G, this has not been described. R6G, in addition to being an acceptor fluorophore, also serves as a model cytostatic drug in drug-release experiments. As one could expect, in aqueous solution, FRET effect was poor, but when exposed to the micelles, both MUTMAC–R6G and FITC–R6G yielded a pronounced FRET effect. Most likely, the formation of micelles is accompanied by the forced convergence of fluorophores in the hydrophobic micelle core by a donor-to-acceptor distance (r) significantly closer than in the aqueous buffer solution, which was reflected in the increase in the FRET efficiency (E). Therefore, r(E) could be used as analytical signal of the micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC), yielding values in good agreement with the literature for similar systems. We found that the r-function provides analytically valuable information about the nature and mechanism of micelle formation. S-S crosslinking between polymer chains makes the micelle more compact and stable in the normal physiological conditions, but loosens in the glutathione-rich tumor microenvironment, which is considered as an efficient approach in targeted drug delivery. Indeed, we found that R6G, as a model cytostatic agent, is released from micelles with initial rate of 5%/h in a normal tissue microenvironment, but in a tumor microenvironment model (10 mM glutathione), the release of R6G from S-S stitched polymeric micelles increased up to 24%/h. Drug-loading capacity differed substantially: from 75–80% for nonstitched polymeric micelles to ~90% for S-S stitched micelles. Therefore, appropriate FRET probes can provide comprehensive information about the micellar system, thus helping to fine-tune the drug delivery system.

FRET Probes as a Technique for Determining the Catalytic Activity of Enzymes in the Model Biomembranes Systems and Further in the Living Cells

“…FRET is observed in aqueous solution for between MUmb-derivative and R6G, but the FRET effect is rather weak, since the donor and acceptor molecules separated from each other in the volume of the solution [7]. A different situation is observed for reverse micellar system, where the enzyme and the reaction components are located in a limited volume of the aqueous phase of the inner cavity of the micelles.…”

“…Previously, we demonstrated that the FRET effect is not as pronounced in aqueous solutions, whereas in micellar systems, this effect is significantly stronger [7]. FRET probes are further applied to study the enzymatic activity in the membrane-like systems such as reverse micelles.…”

“…The paper is devoted to the study of the potential application of the effect of fluorescent resonant energy transfer (FRET [1][2][3][4][5][6][7][8][9][10][11][12][13][14]) as a selective indicator for various biochemical phenomena, in particular the catalytic activity of enzymes in the model biomembrane systems, with an eye on the enzyme activity registration in living cells. Micellar enzymology is an interesting field for the application of FRET [15][16][17].…”

Research Progress of Disulfide Bond Based Tumor Microenvironment Targeted Drug Delivery System

“… 28 Reducing agents such as GSH, dithiothreitol (DTT), and β-mercaptoethanol can react with disulfide bonds, converting them into thiol groups. 29 , 30 The thiol group on cysteine in GSH serves as its active center. Through redox reactions and nucleophilic substitution reactions, disulfide bonds are broken down into thiol groups by GSH, leading to the rapid degradation of the carrier and facilitating drug release for therapeutic effects.…”